User:Wayne Decatur/Haloarcula Large Ribosomal Subunit



The Haloarcula Large Ribosomal Subunit The molecular machine catalyzing petide bond synthesis is a ribozyme.

Introduction
The ribosome is a complex composed of RNA and protein that adds up to several million daltons in size and plays a critical role in the process of decoding the genetic information stored in the genome into protein as outlined in what is now known as the Central Dogma of Molecular Biology. Specifically, the ribosome carries out the process of translation, decoding the genetic information encoded in messenger RNA, one amino acid at a time, into newly synthesized polypeptide chains. The ribosome functions as a complex of two complexes of many proteins and RNAs of substantial length; these two complexes are the small ribosomal subunit and the large ribosomal subunit. The formation of peptide bonds occurs in the large subunit where the acceptor-stems of the tRNAs are docked.

In 2000, the large ribosomal subunit from Haloracula marismortui was solved. Haloracula is a halophilic archaea. The structure revealed that surprisingly no protein was observed close enough the site of peptide bond synthesis to be be involved in the chemistry of the peptidyl transferase reaction, meaning that RNA was responsible for catalysis and that the large subunit is a ribozyme. The structure also revealed the details of the tunnel which the nascent peptide chain would exit the ribosome.

Thomas Steitz shares a 2009 Nobel Prize for The Haloarcula Large Ribosomal Subunit Structure
For this landmark structure, Thomas A. Steitz of Yale University shared the 2009 Nobel Prize in Chemistry along with two other structural biologists working on the ribosome. It is important to note that the Steitz lab worked with the Moore lab on this phenomenal accomplishment although the Nobel committee limits the award itself to up to three laureates. This structure ranks among the known structures with highest impact.

Haloracula Large Ribosomal Subunit Components
The large subunit of the Haloracula marismortui ribosome sediments at 50S, as do the large subunits of archaea and eubacteria. It is composed of two chains of RNA, a 23S chain (2,922 nucleotides long, 946 kDa) and a 5S chain (122 bases long, 39 kDa). Assembled with the RNA are 27 protein chains (of a total of 31 known), varying in length from 49 (L39E, 6 kDa) to 337 amino acids (L3, 37 kDa).

The Haloarcula large ribosomal subunit at first glance:

 * The solved large subunit is a monolith.
 * With no significant portion of the 50S subunit appearing topologically separate or capable of forming a stable structure on its own, the solved structure of the 1.6-million Dalton large subunit is one massive domain .
 * On the other hand, the large subunit's partner in translation, the small subunit (30S), clearly has three domains.
 * It is important to note that two stalks (the L1 stalk and L7/L12 stalk) seen in lower resolution structures on each side of the large ribosomal subunit at lower resolution are not visible in the higher resolution structure viewed here. Thus, in actuality the Haloarcula large subunit has a less monolithic appearance with other protuberances on either side of the central one, yet clearly not possessing the distinct domains formed by the distinct rRNA domains visible in the secondary structure (see below).


 * The large ribosomal subunit is a ribonucleo protein  macromolecule.
 * This macromolecule is mostly sprinkled with several proteins .
 * 27 of the 31 known large subunit  proteins  are visible in the crystal structure. L1, which would be at the 'L1 stalk', is one of the proteins not seen.


 * This macromolecule is made of two  chains:
 * a small 5S rRNA (122 nucleotides)which forms part of the central protuberance seen in the large subunit.
 * a large 23S rRNA (3045 nucleotides) - 2833 of the 3045 nucleotides of the 23S rRNA are seen in the structure.

The rRNA domains:
The secondary structure map of Haloarcula 23S rRNA (below) clearly shows six large RNA domains extending off a large major loop.


 * The six domains of the large subunit ribosomal RNA and 5S rRNA fit into the monolithic subunit like puzzle pieces :
 * Domain I (shown in blue )
 * Domain II ( shown in cyan )
 * Domain III ( shown in yellow )
 * Domain IV (<font color = "#00FF00">shown in green )
 * Helix 69 is a portion of Domain IV and is part of one of the important conserved intersubunit bridges of the ribosome (b2a), although it is not visible in this structure. (The 13-nt stem-loop not seen would connect the highlighted spheres <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/1s72rrnadomain4h69/2'>here .) See the ribosome to see a structure where helix 69 is observed in the solved structure, extending from the large subunit under the A- and P- site tRNAs in the 70S ribosome and contacting the tRNAs and the small subunit decoding center. Helix 69 plays a roles in initiation, termination, and disassembly of the ribosome post-termination.
 * <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/1s72rrnadomain5/6'>Domain V ( shown in red )
 * Domain V lies at the core of the subunit.  It is known to be intimately associated with the peptidyl transferase reaction that occurs during translation. This is further explored further below.
 * <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/1s72rrnadomain6/6'>Domain VI (<font color = "#ae00fe">shown in purple )
 * <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/1s725srrns/3'>5S rRNA (<font color = "#FF00FF">shown in magenta ), though a separate molecule, is effectively the seventh RNA domain of the large subunit.

The proteins:

 * <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/1s72extenvsglb/6'>Globular vs. extended proteins
 * Proteins are generally globular.  However, while about half the 27 proteins seen in the crystal structure of the large ribosomal subunit are globular ( shown as orange ), interestingly, the other half are extended or have large extended regions emanating from globular domains ( shown as cyan ).
 * These extended proteins and regions are reminiscent of the intrinsically unfolded proteins that play roles in many other processes.


 * <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/4examples/12'>Particular examples of globular vs. extended proteins
 * Zooming in to see some examples in more detail:
 * L2, L15, and L39e illustrate proteins with extended regions.
 * For contrast, the globular L7ae is shown. The extended regions are highlighted in cyan.
 * L39e is extended over its entire length.


 * <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/Extensionpenetrate/5'>Extensions penetrate into the subunit interior
 * View of L2, L15, L39e and L7ae again but with the RNA backbone shown. The extended regions are again highlighted in cyan.
 * Using the mouse to spin around the structure clearly shows that the extended proteins penetrate into the interior to fill gaps between RNA secondary structure elements.
 * The globular domains are the portions of the proteins on the subunit's exterior, nestling in the gaps and crevices of the folded RNA. You may need to use the mouse to move the structure around to convince yourself.
 * This view is also a good point to note the fact that the ribosomal proteins do not encase the nucleic acid as with spherical viruses or with Tobacco mosaic virus, nor do the proteins become surrounded by the nucleic acid as in the nucleosome.

The ribosome is a ribozyme - protein DOES NOT participate directly in the chemistry of peptide bond synthesis:

 * <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/1s72rrnadomain5allwithyarus/9'>An informative analog is observed at the core of the large subunit bound to Domain V ( shown in red )
 * In addition to unliganded subunit, the large subunit structure has been solved with substrate analogs which provides a detailed view of the direct role domain V plays in the chemistry of peptide bond synthesis. One of the analogs was the Yarus analog (CCA-puromycin), known to inhibit translation because it mimics normal substrate, specifically it resembles an unstable transition state intermediate formed during peptide bond synthesis and involving the extreme ends of the A- and P- tRNAs and atoms corresponding to parts of an amino acid. <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/1s72rrnadomain5withyarus/4'>The Yarus analog (magenta), indicating the site of the petidyl transferase reaction, is entrenched in Domain V.
 * <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/1s72rrnadomain5withyarusnts/5'>Guanosine 2284 (orange) and Guanosine 2285 (blue) are base-paired with the Yarus analog.
 * <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/1s722486yarus/1'>One atom in particular corresponds to the carbon of the tetrahedral carbon intermediate of the peptidyl transferase reaction, and A2486 (E. coli #2451) of Domain V approaches this critical atom of the Yarus analog . The individual components of the Yarus analog are labeled.
 * It is important to note that the ribosome is highly conserved, particularly the nucleotides close to the Yarus analog in Domain V and thus the major conclusions reached from the structure are applicable to all ribosomes.
 * <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/1s72ribozyme/13'>No proteins approach close enough to the active site to affect the chemistry of peptide bond synthesis
 * L2, L3 , <font color="#ae00fe">L4 and L10e are the nearest proteins to the Yarus analog bound to domain V of the subunit. Note that it may help to start from <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/1s72beststartplusanalog/1'> the entire subunit with analog bound before hitting the above <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/1s72ribozyme/13'>scene link to get a good sense of these elements in the core of the subunit or use <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/1s72ribozymewall/3'>this scene with the rest of the subunit shown translucently . With <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/1s72ribozymewp/2'>all the proteins shown it is obvious these four proteins are the closest.
 * Examining the distance of the proteins from the phosphorous analog of the tetrahedral carbon ( in yellow ) indicates none of the proteins are close enough to be involved in the chemistry of peptide bond synthesis. Even the closest protein is over 15 &Aring; away. (In the eubacterial ribosomes, e.g., 2j01,2i2v, and 2wdn, the N-terminus of a non-universally conserved protein, L27, also comes close to the active site  .)
 * As touched on earlier in this section as well as in an earlier section, it is in fact, <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/1s72ribozymewprna/2'>the RNA of Domain V that is intimately associated with the active site of the ribosome, leaving little doubt that the ribosome is indeed a ribozyme.
 * Keep in mind that in order to make this large molecule load in reasonable times over the internet, the RNA has been simplified by leaving out the information for most of the bases and in fact if all the bases were included it would look even more crowded with RNA at the active site.
 * SUMMARY: Only RNA is in proximity to the site of peptide bond synthesis, and therefore the chemistry of peptide bond synthesis is not catalyzed by protein and in fact the ribosome is a ribozyme with the peptidyl transferase reaction being catalyzed by RNA.

Polypeptide Exit Tunnel:

 * <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/1s72tunnelend/2'>The end of the Polypeptide Exit Tunnel is shown in the center
 * As the nascent chain grows, it advances into a tunnel about 100 angstroms long that passes through the large subunit, called the polypeptide exit tunnel.
 * The diameter averages about 15&Aring;; at no point is the diameter within the tunnel big enough to accommodate any folding of the nascent chain to a greater extent than alpha-helices.
 * The wall of the tunnel is mostly RNA (ca. 80%) and the proteins are shown translucently to emphasize this point and for clarity.
 * <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/1s72tunnell22l4/4'>L22 and L4 proteins form part of the tunnel wall, making a <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/1s72tunnell22l4/3'>constriction about midway , and have been suggested to play roles in regulation of translation    , particularly in the case of L22, although an involved portion is dispensable for growth.
 * <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/1s72tunnelendpro/3'>Proteins L19, L22, L23, L24, L37e, and L39e encircle this opening which is where the nascent polypeptide chain will first emerge from the ribosome. Here the nascent chain can contact chaperones, such as trigger factor, and the machinery for transferring the growing chain across a membrane, the signal-recognition particle.


 * <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/1s72tunnelstart/2'>The start of the Polypeptide Exit Tunnel tunnel
 * Each amino acid added to the growing peptide chain will begin its journey through the tunnel at this end.
 * The <scene name='User:Wayne_Decatur/Sandbox_Haloarcula_Ribosomal_Large_Subunit/1s72tunnelstartyarus/4'>start of the tunnel corresponds to the site where the Yarus analog (magenta) binds in the active site. This makes sense since the analog represents the intermediate in peptide bond synthesis.
 * A number of antibiotics, including the macrolide Azithromycin (zithromax), binds to this region of the tunnel.

Structures
Steitz and Moore labs original atomic-resolution structures : Haloarcula marismortui large ribosomal subunit - 1ffk and later refined to give 1jj2, and then refined to give 1s72 , 2qa4 , and later 3cc2. Related: 1ffz, 1fg0. Assembled with the ribosomal RNAs (2,922 and 122 nucleotides long) in the structure are 27 protein chains (of a total of 31 known), varying in length from 49 (L39E, 6 kDa) to 337 amino acids (L3, 37 kDa). Specifically used on this page were 1s72 with Yarus analog interacting nucleotides from 1ffz.

External Resources

 * 70S Ribosome: January 2010 Molecule of the Month as part of the series of tutorials that are at the RCSB Protein Data Bank and written by David Goodsell
 * RCSB Protein Data Bank coverage of the 2009 Nobel Prizes in Chemistry
 * The Nobel Prize in Chemistry 2009 page at The Official Web Site of the Nobel Prize
 * Ribosome: October 2000 Molecule of the Month as part of the series of tutorials that are at the RCSB Protein Data Bank and written by David Goodsell
 * A detailed secondary structure of Haloarcula marismortui 23S rRNA at the 3D Ribosomal Modifications Map Database